extraction-and-dissection-of-the-domesticated-pig-brain

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

University of Illinois at Urbana-Champaign

The underlying neuronal mechanisms of learning and memory have been heavily explored using associative learning paradigms. Two of the more commonly employed learning paradigms have been contextual and delay fear conditioning. In fear conditioning, a subject learns to associate a neutral stimulus (conditioned stimulus; CS), such as a tone or the context of the room, with a fear provoking stimulus (unconditioned stimulus; US), such as a mild footshock. Utilizing these two paradigms, various analyses have elegantly demonstrated that the amygdala plays a role in both fear-related associative learning paradigms. However, the amygdala's involvement in trace fear conditioning, a forebrain-dependent fear associative learning paradigm that has been suggested to tap into higher cognitive processes, has not been closely investigated. Furthermore, to our knowledge, the specific amygdala nuclei involved with trace fear conditioning has not been examined. The present study used Arc expression as an activity marker to determine the amygdala's involvement in trace fear associative learning and to further explore involvement of specific amygdalar nuclei. Arc is an immediate early gene that has been shown to be associated with neuronal activation and is believed to be necessary for neuronal plasticity. Findings from the present study demonstrated that trace-conditioned mice, compared to backward-conditioned (stimulation-control), delay-conditioned and naïve mice, exhibited elevated amygdalar Arc expression in the basolateral (BLA) but not the central (CeA) or the lateral amygdala (LA). These findings are consistent with previous reports demonstrating that the amygdala plays a critical role in trace conditioning. Furthermore, these findings parallel studies demonstrating hippocampal-BLA activation following contextual fear conditioning, suggesting that trace fear conditioning and contextual fear conditioning may involve similar amygdala nuclei. Together, findings from this study demonstrate similarities in the pathway for trace and contextual fear conditioning, and further suggest possible underlying mechanisms for acquisition and consolidation of these two types of fear-related learning. 

Elevated Arc/Arg 3.1 protein expression in the basolateral amygdala following auditory trace-cued fear conditioning.

Novelty preference paradigms have been widely used to study recognition memory and its neural substrates. The piglet model continues to advance the study of neurodevelopment, and as such, tasks that use novelty preference will serve especially useful due to their translatable nature to humans. However, there has been little use of this behavioral paradigm in the pig, and previous studies using the novel object recognition paradigm in piglets have yielded inconsistent results. The current study was conducted to determine if piglets were capable of displaying a novelty preference. Herein a series of experiments were conducted using novel object recognition or location in 3- and 4-week-old piglets. In the novel object recognition task, piglets were able to discriminate between novel and sample objects after delays of 2min, 1h, 1 day, and 2 days (all P<0.039) at both ages. Performance was sex-dependent, as females could perform both 1- and 2-day delays (P<0.036) and males could perform the 2-day delay (P=0.008) but not the 1-day delay (P=0.347). Furthermore, 4-week-old piglets and females tended to exhibit greater exploratory behavior compared with males. Such performance did not extend to novel location recognition tasks, as piglets were only able to discriminate between novel and sample locations after a short delay (P>0.046). In conclusion, this study determined that piglets are able to perform the novel object and location recognition tasks at 3-to-4 weeks of age, however performance was dependent on sex, age, and delay.

Young pigs exhibit differential exploratory behavior during novelty preference tasks in response to age, sex, and delay.

Sialic acid (SA) is a key component of gangliosides and neural cell adhesion molecules important during neurodevelopment. Human milk contains SA in the form of sialyllactose (SL) an abundant oligosaccharide. To better understand the potential role of dietary SL on neurodevelopment, the effects of varying doses of dietary SL on brain SA content and neuroimaging markers of development were assessed in a newborn piglet model. Thirty-eight male pigs were provided one of four experimental diets from 2 to 32 days of age. Diets were formulated to contain: 0 mg SL/L (CON), 130 mg SL/L (LOW), 380 mg SL/L (MOD) or 760 mg SL/L (HIGH). At 32 or 33 days of age, all pigs were subjected to magnetic resonance imaging (MRI) to assess brain development. After MRI, pig serum and brains were collected and total, free and bound SA was analyzed. Results from this study indicate dietary SL influenced ( = 0.05) bound SA in the prefrontal cortex and the ratio of free SA to bound SA in the hippocampus ( = 0.04). Diffusion tensor imaging indicated treatment effects in mean ( 0.01), axial ( 0.01) and radial ( = 0.01) diffusivity in the corpus callosum. Tract-based spatial statistics (TBSS) indicated differences ( 0.05) in white matter tracts and voxel-based morphometry (VBM) indicated differences ( 0.05) in grey matter between LOW and MOD pigs. CONT and HIGH pigs were not included in the TBSS and VBM assessments. These findings suggest the corpus callosum, prefrontal cortex and hippocampus may be differentially sensitive to dietary SL supplementation.

Dietary Sialyllactose Influences Sialic Acid Concentrations in the Prefrontal Cortex and Magnetic Resonance Imaging Measures in Corpus Callosum of Young Pigs.

Sialic acid (SA) is an integral component of gangliosides and signaling molecules in the brain and its dietary intake may support cognitive development. We previously reported that feeding sialyllactose, a milk oligosaccharide that contains SA, alters SA content and diffusivity in the pig brain. The present research sought to expand upon such results and describe the effects of feeding sialyllactose on recognition memory and sleep/wake activity using a translational pig model. Pigs were provided ad libitum access to a customized milk replacer containing 0 g/L or 380 g/L of sialyllactose from postnatal day (PND) 2-22. Beginning on PND 15, pigs were fitted with accelerometers to track home-cage activity and testing on the novel object recognition task began at PND 17. There were no significant effects of diet on average daily body weight gain, average daily milk intake, or the gain-to-feed ratio during the study (all  ≥ 0.11). Pigs on both diets were able to display recognition memory on the novel object recognition task ( < 0.01), but performance and exploratory behavior did not differ between groups (all  ≥ 0.11). Total activity and percent time spent sleeping were equivalent between groups during both day and night cycles (all  ≥ 0.56). Dietary sialyllactose did not alter growth performance of young pigs, and there was no evidence that providing SA via sialyllactose benefits the development of recognition memory or gross sleep-related behaviors.

Dietary Sialyllactose Does Not Influence Measures of Recognition Memory or Diurnal Activity in the Young Pig.

Human milk contains a unique profile of oligosaccharides (OS) and preliminary evidence suggests they impact brain development. The objective of this study was to assess the impact of bovine and/or human milk oligosaccharides (HMO) (2'-fucosyllactose and Lacto-N-neotetraose) on cognition, brain development, and hippocampal gene expression. Beginning on postnatal day (PND) 2, male pigs received one of four milk replacers containing bovine milk oligosaccharides (BMOS), HMO, both (BMOS + HMO), or neither. Pigs were tested on the novel object recognition task using delays of 1- or 48-h at PND 22. At PND 32-33, magnetic resonance imaging procedures were used to assess structural brain development and hippocampal tissue was collected for analysis of mRNA expression. Pigs consuming only HMO exhibited recognition memory after a 1-h delay and those consuming BMOS + HMO exhibited recognition memory after a 48-h delay. Both absolute and relative volumes of cortical and subcortical brain regions were altered by diet. Hippocampal mRNA expression of , , , and  were most strongly affected by diet. HMO and BMOS had distinct effects on brain structure and cognitive performance. These data suggest different mechanisms underlie their influence on brain development.

Human and Bovine Milk Oligosaccharides Elicit Improved Recognition Memory Concurrent With Alterations in Regional Brain Volumes and Hippocampal mRNA Expression.

Article	Year
Elevated Arc/Arg 3.1 protein expression in the basolateral amygdala following auditory trace-cued fear conditioning. Neurobiology of learning and memory\| PubMed ID: 23891993
Young pigs exhibit differential exploratory behavior during novelty preference tasks in response to age, sex, and delay. Behavioural brain research\| PubMed ID: 28042005	2017
Dietary Sialyllactose Influences Sialic Acid Concentrations in the Prefrontal Cortex and Magnetic Resonance Imaging Measures in Corpus Callosum of Young Pigs. Nutrients\| PubMed ID: 29182578	2017
Dietary Sialyllactose Does Not Influence Measures of Recognition Memory or Diurnal Activity in the Young Pig. Nutrients\| PubMed ID: 29570610	2018
Human and Bovine Milk Oligosaccharides Elicit Improved Recognition Memory Concurrent With Alterations in Regional Brain Volumes and Hippocampal mRNA Expression. Frontiers in neuroscience\| PubMed ID: 32903658	2020

Stephen A. Fleming

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